1. Field of the Invention
This invention relates to pharmaceutical compositions comprising antibodies or fragments thereof which are specific for Stem Cell Factor (SCF), and methods for testing and using such pharmaceutical compositions in a defined dose range to alleviate asthma and related inflammatory disease in mammals.
2. Description of the Prior Art
Asthma is a lung disease characterized by (1) airways obstruction that is reversible (but not completely in some patients), either spontaneously or with treatments, (2) airways inflammation, and (3) increased airways responsiveness to a variety of stimuli. Asthma involves airways obstruction which is due to a combination of factors that include (1) spasm of airways smooth muscle; (2) edema of airways mucosa; (3) increased mucus secretion; (4) cellular, especially eosinophilic, infiltration of the airways walls; and (5) injury and desquamation of the airways epithelium. (see generally The Merck Manual of Diagnosis and Therapy, 7th edition, Berkow, R. ed., Merck Research Laboratories, Rahway, N.J. 1992, chp. 34). About 10 million asthmatics live in the United States. From 1980 to 1987 there has been a 29% increase in the prevalence rates of asthma, and from 1970 to 1987 hospital discharge rates for asthma nearly tripled. More alarmingly, the mortality rate from asthma worldwide is increasing, in the United States alone increasing 37% from 1980 to 1987.
The alleviation or prevention of asthma related inflammation is highly desirable, for both human reasons and as an adjunct to proper effective clinical management of asthmatic disease. Aside from prophylactic steps to minimize exposure to certain environmental factors, and nonspecific exacerbating factors, treatment of asthma is conveniently considered as management of the acute attack and day-to-day therapy. Conventional methods of treatment can be classified under five groups of drug therapies: .beta.-adrenergic agents, theophylline, corticosteroids, cromolyn sodium and anticholinergic agents. (M. Windt, 1991, "Ashma: Evolving Therapeutic Regimens", SPECTRUM Phamaceuticals, Decision Resources, Inc., pp23-1 to 23-7).
.beta.-Adrenergic agents (.beta.1 and .beta.2) are useful for treating acute asthma attacks, or to prevent acute attack after exercise, because of their rapid onset (usually within minutes),and relatively short duration (4 to 6 hours at most). It is believed that unfortunately, treatment with .beta.2-adrenergic agents alone, may in fact potentiate hyperresponsiveness and thereby make patients more susceptible to asthma attack. (C. P. Page, 1993, "An Explanation of the Asthma Paradox", Am Rev Respir Dis 147:S29-S32). Adverse effects are more common after oral administration than aerosol, because of the general effects on smooth and cardiac muscles. Theophylline (a methylxanthine) also relaxes smooth muscle, but does not inhibit mediator release by granulocyte, nor does it inhibit hyperresponsiveness following antigen challenge or long-term use. Corticosteroids are general anti-inflammatory agents which inhibit the attraction of polymorphonuclear leukocytes to the site of allergic reaction. Long term therapy can decrease bronchial hyperresponsiveness (especially after aerosol administration). Systemic corticosteroids are very effective, but are reserved for more difficult acute episodes because of the many potential adverse effects. Cromolyn sodium (DSCG, disodium cromoglycate), used prophylactically appears useful in treating children and some adults, as maintenance therapy. It is not suitable for treating acute attacks. In the United States, cost and problems with patient compliance has limited its use, even though this is the safest drug available for the treatment of asthma. Anti-cholinergic agents block cholinergic pathways that cause airway obstruction and may provide additional bronchodilator effect in treating acute attack. Such methods are unsatisfactory because they do not clinically address the source of the inflammatory response, the physiological problem which leads to asthmatic sensitivity, and because of the side effects of such powerful drugs on the patient.
The pathophysiology of asthma is marked by an inflammatory state in which the airways are narrowed chronically by edema and episodically by a variety of spasmogens that are released from resident and infiltrating cells. (see generally Goodman and Gilman's--The Pharmacological Basis of Therapeutics, 8th edition, Gilman, Rall, Nies, and Taylor eds., Pergamon Press, New York, N.Y., 1990; chp. 25). Asthmatic hyperresponsiveness to chemical and physical stimuli can cause bronchoconstriction almost immediately (immediate response), as well as an episode several hours later (late response). This late response appears to occur due to the recruitment of circulating cells which are attracted to the area by chemotactic factors (mediators), which serve to maintain or intensify the inflammatory state. It is believed that environmental antigens are responsible for maintaining a state of hyperresponsiveness which underlies the asthma of many individuals. The list of known or suspected mediators which may play a role in the allergic responses of asthma have been growing steadily over the years and currently include histamine, prostanoids, leukotrienes, and platelet-activating factor (PAF). Also implicated are the release of acetylcholine and neuropeptides such as substance P from parasympathetic nerves.
Generally, the focus of therapeutic intervention of the mechanisms of asthma has focused on inhibition or antagonism of chemical mediators involved in cell recruitment, potentiation, and maintenance of the hyperresponsive state. Such therapies aim to reduce the activation or accumulation of inflammatory cells in the sensitive regions. (J. Morley, 1993, "Immunopharmacology of asthma", TiPS 14:208-213). As appreciation of the complexity of the pathophysiology of asthma has increased, certainty as to the mechanism of action and the cellular targets of therapeutic agents has decreased. This is a major limitation to the use of mediator based pharmaceuticals for treating asthma, which is in addition to the unwanted side effects associated with such treatments. Atopy has become a focus for some researchers, and directs pharmacological intervention of mediators and cells on the immunological/immunogenic basis of asthma. (R. Djkanovic et al., 1993, "Mechanisms of airways inflammation which may be amenable to prophylaxis", AAS Update on Childhood Asthma 40:169-180). Many cytokines have been studied with the goal of determining the cell sources. (V. Ackerman et al., 1994, "Detection of cytokines and their cell sources in bronchial biopsy specimens from asthmatic patients", Chest 105:687-696). The predominant cells bearing IgE in the late-phase response to antigen in the lung was identified as being basophils in one study focusing on histamine as a key mediator. (C. B. Guo et al., 1994, "Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils", Am J Respir Cell Mol Biol 10:384-390). Thus, while the focus is still on reduction of hyperreactivity, and reducing inflammation by intervention with the mediator signals, it has been recognized that a reduction in the number of reactive cells will apparently result in reduced inflammation. (S. Godfrey, 1993, "Airway inflammation, bronchial reactivity and asthma", AAS Update on Childhood Asthma 40:109-143).
It would, therefore, be advantageous to be able to utilize a pharmaceutical composition and a treatment which produce minimal toxicity or side effects, but provide highly effective, reproducible results yielding a reduction in the number of reactive cells, thereby avoiding the complexity and confusion over therapeutic intervention with mediator signals, by preventing the action of the responsible cells.
It has been shown that stem cell factor (SCF, c-kit ligand, steel factor) critically regulates the migration and survival of mast cell precursors, promotes the proliferation of both immature and mature mast cells, enhances mast cell maturation, directly induces secretion of mast cell mediators, and can regulate the extent of mediator release in mast cells activated by IgE-dependant mechanisms. (S. J. Galli et al., 1993, "The c-kit Receptor, Stem Cell Factor, and Mast Cells", Am J Pathology 142:965-974). In addition, SCF is one of the hematopoietic growth factors are a family of glycoproteins involved in the production of blood cells from bone marrow precursors. (K. Kaushansky, 1992, "Structure-function relationships of the hematopoietic growth factors", Proteins: Structure, Function, and Genetics 12:1-9). SCF appears to also be a costimulatory factor in many of the hematopoietic cell lines, but required multi-lineage factors such as IL-3 in order to maintain their development. (M-L Li et al., 1993, "Co-stimulatory effects of steel factor; the c-kit ligand, on purified human hematopoietic progenitors in low density cell culture", Nouv Rev Fr Hematologie 35:81-86; H. E. Broxmeyer et al., 1991, The Kit receptor and its ligand, steel factor, as regulators of hematopoiesis", Cancer Cells 3:480-487; H. Saito et al., 1994, "Growth in methylcellulose of human mast cells in hematopoietic colonies stimulated by steel factor, a c-kit ligand", int Arch Allergy Immunol 103:143-151). The action of Kit ligand and IL-3 may control the synthesis of serotonin in mast cells. (I. Ziegler et al, 1993, "In a concerted action Kit ligand and interleukin 3 control the synthesis of serotonin in murine bone marrow-derived mast cells", J Biological Chem 268:12544-12551). Interestingly enough, SCF/KIT has been found associated with neurological pathologies as well. (I. J. Ryan et al., 1994, "Role for the Stem Cell Factor/KIT complex in Schwann cell neoplasia and mast cell proliferation associated with neurofibromatosis", J Neurosci Res 37:415-432).
It has been found that both murine and human recombinant c-kit ligands are active on human mast cell precursors, indicating that the structure and function is highly conserved if not essentially identical. (A. M. Dvorak et al., 1993, "Human and murine recombinant c-kit ligands support the development of human mast cells from umbilical cord blood cells: ultrastructural identification", Int Arch Allergy Immunol 101:247-253).
It appears that SCF has emerged as the key to the riddle of mast cell activity. (P. Valent, 1994, "The riddle of the mast cell: kit(CD-117)-ligand as the missing link?", Immunology Today 15:111-114). Recent work has shown that there is localization of the receptor for SCF (c-kit product, KIT) in various tissues. (Y. Tsuura et al., 1994, "Preferential localization of c-kit product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: immunohistochemical study of formalin-fixed, paraffin-embedded tissues", Virchows Archiv 424:135-141).
It has been found that the c-kit ligand promotes mast cell survival by suppressing apoptosis. (A. Iemura et al., 1994, "The c-kit ligand, Stem Cell Factor, promotes mast cell survival by suppressing apoptosis", Am J Pathology 144:321-328). Treatment with rhSCF (recombinant human SCF) was found to reversibly expand mast cell populations in primates in vivo. (S. J. Galli et al, 1993, "Reversible expansion of primate mast cell populations in vivo by stem cell factor", J Clin Invest 91:148-152). SCF and c-kit have been found to regulate cell-matrix adhesion to fibronectin in a transient manner. (T. Kinashi and T. A. Springer, 1994, "Steel factor and c-kit regulate cell-matrix adhesion", Blood 83:1033-1038). This adhesion was not dependent on IL-3 and was blocked by antibody to SCF. (J. Dastych and D. D. Metcalfe, 1994, "Stem Cell Factor induces mast cell adhesion to fibronectin", J Immunology 152:213-219). Yet final differentiation of mast cell may require interaction with fibroblasts. (A. M. Dvorak et al.,1993, "Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; distinction from mature basophillic leukocytes undergoing secretion in the same cultures", J Leukocyte Bio 54:465-485). SCF in the presence of IL-3 increases the ratio of mast cells to basophils and alters the ultrastructural characteristics of mast cells and basophils toward a more mature phenotype. (M. Rottem et al, 1993, J Immunology 151:4950-4963).
Work has suggested that rhSCF and anti-IgE may act on human mast cells through a common pathway to increase free cystolic calcium, and that this effect can be similarly modulated by various drugs. (M. Columbo et al., 1994, "Studies of the intracellular Ca2+ levels in human adult skin mast cells activated by the ligand for the human c-kit receptor and anti-IgE", Biochem Pharmacol 47:2137-2145). Other work has shown that mast cells potentiate the release of cytokines in response to IgE cross-linking, and perhaps does not act to stimulate release directly. (S. C. Bischoff and C. A. Dahinden, 1992, "c-kit ligand: a unique potentiator of mediator release by human lung mast cells", J Exp Med 175:237-244). In contrast, work with rrSCF (recombinant rat SCF) in mice indicates that chronic treatment with rrSCF induced mast cell hyperplasia, but does not increase the severity of IgE-dependent anaphylactic reactions. (A. Ando et al, 1993, "Effects of chronic treatment with the c-kit ligand, Stem Cell Factor, on immunoglobulin E-dependent anaphylaxis in mice", J Clin Invest 92:1639-1649).
There have been several patent publications in relation to SCF and c-kit product the SCF receptor.
European patent application 0 548 867 A2, published 30.06.93, teaches a soluble stem cell factor (SCF)-receptor (KIT, c-kit product).
International patent application PCT/US 92/02674, published Oct. 15, 1992, teaches monoclonal antibodies to stem cell factor receptors.
International patent application PCT/US 91/06130, published Mar. 5, 1992, teaches a ligand for the c-kit receptor and methods of use.
UK patent application GB 2 258 234 A, published Feb. 3, 1992, teaches a soluble Kit ligand (KL) protein.
European patent application 0 423 980 A1, published Apr. 24, 1991, teaches stem cell factor and methods of use for treating blood disorders.
International patent application PCT/US 93/03640, published Nov. 11, 1993, teaches ligand for the c-kit receptor and methods of use thereof.
There remains a need in the medical arts for an effective treatment for asthma, and the inflammation that is associated with asthma. Although its importance and use for maintaining cell cultures of hematopoietic stem cells and therapeutic uses in stimulating hematopoiesis is known, SCF has not been shown to be a desirable or effective target for any therapeutic intervention in the treatment of asthma.
As discussed earlier, the pathophysiology of asthma is not clear. The study of the pathophysiology has been hampered by the lack of affordable, uniform, and well characterized animal models for the study of atopic asthma. Atopic asthma in humans has been described as an allergic disease characterized by reversible obstruction of the airways or bronchi. The immune response associated with the onset of asthma has been described as having mixed histopathological features of both acute and chronic, cell-mediated immune reactions. This response is histopathologically characterized by the infiltration of the bronchial mucosa with neutrophils, eosinophils, macrophages, and lymphocytes (see for review C. J. Corrigan and A. B. Kay, 1992, "T cells and eosinophils in the pathogenesis of asthma", Immunol Today 13:501-507), though basophils have been indicated as being a source of chemotactic factors. Data on asthma suggests that the onset of the asthmatic response is controlled by CD4+ T-lymphocytes which produce a characteristic TH2 pattern of lymphokine production (A. B. Kay and C. J. Corrigan, 1992, "Asthma, eosinophils and neutrophils", Br Med Bull 48:51-64). The TH2 pattern of lymphokines consists of expression of IL-4, IL-5, and IL-10 (Mossman and Moore, 1989, "The role of IL-10 in crossregulation of Th1 and Th2 response", Immunol Today 12:A49-A58; Mossmann, T. R. et al., 1986, "Two types of murine helper T cell clone", J Immunol 136:2348-2359). The expression of these lymphokines correlates well with asthma as their individual functions play a role in the asthmatic response. The expression of IgE (IL-4) (Zhang, X. et al., 1992, "T cells from atopic individuals produce IgE-inducing activity incompletely blocked by anti-interleukin-4 antibody", Eur J Immunol 22:829-833) and eosinophilia (IL-4/IL-5) (Spry, C. J. et al., 1992, "Eosinophils", Immunol Today 13:384-387) are both characteristic of asthmatic responses (Del Prete, G., 1992, "Human Th1 and Th2 lymphocytes: their role in the pathophysiology of atopy", Allergy 47:450-455). However, a primary trait of asthma is the accumulation of eosinophils in the bronchoalveolar lavage (BAL) fluid (Corrigan and Kay, 1992; Kay and Corrigan, 1992; Arm, J. P. and T. H. Lee, 1992, "The pathobiology of bronchial asthma", Adv Immunol 51:323-382; Diaz et al., 1989, "Leukocytes and mediators in bronchoalveolar lavage during allergen-induced late-phase asthmatic reactions", Am Rev Respir Dis 139:1383-1389). Historically, eosinophils have been implicated as a primary cell responsible for the induction of bronchial mucosal injury and are thought to induce the bronchial obstruction associated with the asthmatic response (Kay and Corrigan, 1992; Djukanovic et al., 1990, "Mucosal inflammation in asthma", Am J Respir Dis 142:434-457; Walker et al., 1993, "Increased expression of CD11b and functional changes in eosinophils after migration across endothelial cell monolayers", J Immunol 150:4061-4071). Due to the difficulty of longitudinal studies of patient populations and paucity of asthma-related data in animal models, the mechanisms which lead to the pathophysiology of this disease are not presently clear. It would be most useful to the medical and scientific community to have a murine model to examine the cellular and molecular events involved in the asthmatic response.
In order to develop and test pharmaceutical treatments for asthma, it would be useful to have an appropriate, characterizable, economical and readily available animal model system for the disease. Laboratories have employed, as animal models, the use of various antigens or pharmaceutical agents in dogs and primates as well as guinea pigs (Mapp et al., 1985, "Airway responsiveness to inhaled antigen, histamine, and methacholine in inbred, ragweed sensitive dogs", Am Rev Respir Dis 132:292-298; Sasaki et at, 1989, "Late asthmatic response to Ascaris antigen challenge in dogs treated with mtyrapone", Am Rev Respir Dis 136:1459-1465; Yamada et al., 1992, "Development of an animal model of late asthmatic response in guinea pigs and effects of anti-asthmatic drugs", Prostaglandins 43:507-521). Work with the canine system has not been greatly expanded, and is hampered by the cost of the animals, care, lack of characterized reagents, ease of generating large numbers of genetically identical individuals, and strict regulations.
At present two animal models are mainly being used to study asthma. The first is an Ascaris suum parasite antigen-induced primate model system (R. H. Gundel et al., 1992, "Antigen-induced acute and late-phase responses in primates", Am Rev Respir Dis 146:369-373; D. I. Pritchard et al., 1983, "Laboratory infection of primates with Ascaris suum to provide a model of allergic bronchoconstriction", Clin Exp Immunol 54:469-476). There have been major obstacles to the use of this model system, like the canine system, the least of which has been limits of strict regulations and prohibitive cost of the animals.
The second animal model being used is induced in guinea pigs by various antigens (H. Iijima et al., 1987, "Bronchoalveolar lavage and histologic characterization of late asthmatic response in guinea pigs", Am Rev Respir Dis 136:922-929; K. Ishida et al, 1989, "Repeated antigen challenge induces airway hyperresponsiveness with tissue eosinophilia in guinea pigs", J Appl Physiol 67:1133-1139; C. Vertes et al., 1987, "A model for experimental asthma: provocation in guinea-pigs immunized with Bordetella perussis", Bull Eur Physiopathol Respir 10:111s-113s), and pharmaceuticals (J. P. Hayes et al , 1992, "Bronchoconstriction and airway microvascular leakage in guinea pigs sensitized with trimellitic anhydride", Am Rev Respir Dis 146:1306-1310; H. Obata et al.,1992, "Guinea pig model of immunologic asthma induced by inhalation of trimellitic anhydride", Am Rev Respir Dis 146:1553-1558). Unfortunately, there are major difficulties with this system, among them are the limited array of reagents available for the examination of leukocyte subsets and cytokines produced during the asthma reaction. Difficulty and cost of acquiring, breeding, and maintaining the animals is also a deterrent to their use.
Thus there is still a need for a useful model, and method for generating such an animal model for asthma which would have the advantages of reagent availability for characterization of the disease, low cost, and the ability for assessing a large number of genetically similar animals during pathogenesis of airway inflammation compatible with asthma.
The normal immune response to parasite eggs (soluble egg antigen; SEA), and secreted antigen, during parasite infection, has previously been demonstrated to induce a TH2 driven response, which includes high IL-4 levels and a striking eosinophilia (Chensue et al., 1992, "Role of IL-4 and IFN-gamma in Schistosoma mansoni egg-induced hypersensitivity granuloma formation", J Immunol 148:900-911; Grzych et al., 1991, "Egg deposition is the major stimulus for the production of Th2 cytokines in murine schistosomiasis mansoni", J Immunol 146:1322-1329). Presumably the nature of SEA predisposes the immune system to a TH2 type response, (Lukacs and Boros, 1992, "Utilization of fractionated soluble egg antigens reveals selectively modulated granulomatous and lymphokine responses during murine schistosomiasis mansoni", Infect Immunol 60:3209-3216; Lukacs and Boros, 1993, "Lymphokine regulation of granuloma formation in murine schistosomiasis mansoni", Clin Immunol Immunopaht 68:57-63), which may be exploited for developing a murine model system for asthma.